Ferritin iron uptake and release. Structure–function relationships

Iron uptake and release by ferritin molecules of different iron contents show similar profiles. These are discussed in relation to the structure of the ferritin molecule. Two models of iron uptake and release are considered. One involves iron oxidation–reduction sites on the protein. The other allows direct interaction of reagents with the iron-core crystallites. It is concluded that the second model accounts better for the experimental results presented now and in previous publications.     

马脾铁蛋白释放铁的反应级数和速率相数的转换

采用差示法研究铁蛋白释放铁的动力学规律和反应级数的转换。结果表明：马脾铁蛋白释放铁的速率及相数与还原剂Ｎａ２Ｓ２Ｏ４浓度及铁还原速率无关,与该蛋白蛋白壳的调节速率有关。在ｐＨ５．０￣６．０范围内,马脾铁蛋白以三相不同速率方式释放占原铁核总铁量８０％的铁。但在ｐＨ９．０介质中,ＯＨ＾－不仅能参与铁蛋白铁核组成,减缓释放铁的速率,而且使原混合级反应转换为一级反应,从而使铁蛋白释放铁的动力学过程由复杂转     

Studies on the mechanism of iron release from transferrin.

Iron release from human, rabbit, rat and sheep transferrin, chicken conalbumin and human lactoferrin was measured by the change in absorbance of solutions of the iron-protein complexes or by the release of 59Fe from the protein conjugated to agarose. Several phosphatic compounds and iron chelators were able to mediate the process (ATP, GTP, 2,3-diphosphoglycerate, inositol hexaphosphate, pyridoxal 5-phosphate, cytidine 5-triphosphate, pyrophosphate, inorganic phosphate, citrate, EDTA, oxalate, nitrilotriacetate). The greatest rate of iron release was found with pyrophosphate and the least with inorganic phosphate. Different rates of iron release were obtained with the different proteins, greatest with human transferrin and least with lactoferrin. With each of the proteins and the mediators there was a linera relationship between the H+ concentration and the rate of iron release. At any given pH the rate of iron release increased to a maximal rate as the mediator concentration was raised. It is concluded that iron release from transferrin under the conditions of these experiments involves an initial interaction between H+ and the iron-transferrin complex followed by release of the iron under the action of the mediator.     

Iron binding by phosvitin: variation of rate of iron release as a function of the degree of saturation of iron binding sites

The rate of iron release from Fe(III)-phosvitin complexes, at varied degrees of saturation, was studied. Iron release was induced by reduction in the presence of the ferrous ion chelator, o-phenanthroline. If iron release was induced photochemically (without a chemical reductant), the reactions proceeded in zero order fashion, independently of the degree of saturation but with a strong dependence on the concentration of phenanthroline. When hydroquinone was added and the reactions were conducted in the dark, iron release followed first-order kinetics and the rate constants showed a clear dependence on the degree of saturation of the protein, which was most marked at lower levels of saturation. The results imply control of iron release by binding site differences produced by different intramolecular environments as the protein provides different combinations of its phosphoserine groups as ligands depending on the number of iron atoms to be accommodated per protein molecule.     

Impact of Surface Active Compounds on Iron Catalyzed Oxidation of Methyl Linolenate in AOT–Water–Hexadecane Systems

Edible oils contain minor surface active components that form micro-heterogeneous environments, such as reverse micelles, which can alter the rate and direction of chemical reactions. However, little is known about the role of these micro-heterogeneous environments on lipid oxidation of bulk oil. Our objective was to evaluate the ability of water, cumene hydroperoxide, oleic acid, and phosphatidylcholine to influence the structure of reverse micelles in a model oil system: sodium bis(2-ethylhexyl) sulfosuccinate (aerosol-OT; AOT) in n-hexadecane. The influence of reverse micelle structure on iron catalyzed lipid oxidation was determined using methyl linolenate as an oxidizable substrate. The size and shape of the reverse micelle were investigated by small-angle x-ray scattering, and water contents was determined by Karl Fischer titrations. Lipid hydroperoxides and thiobarbituric acid reactive substances were used to follow lipid oxidation. Our results showed that AOT formed spherical reverse micelles in hexadecane. The size of the reverse micelles increased with increased water or phosphatidylcholine concentration, but decreased upon addition of cumene hydroperoxide or oleic acid. Iron catalyzed oxidation of methyl linolenate in the reverse micelle system decreased with increasing water concentration. Addition of phosphatidylcholine into the reverse micelle systems decreased methyl linolenate oxidation compared to control and reverse micelles with added oleic acid. These results indicate that water, cumene hydroperoxide, oleic acid, and phosphatidylcholine can alter reverse micelle size and lipid oxidation rates. Understanding how these compounds influence reverse micelle structure and lipid oxidation rates could provide information on how to modify bulk oil systems to increase oxidative stability.     

Anion-mediated iron release from transferrins. The kinetic and mechanistic model for N-lobe of ovotransferrin

Iron release process of ovotransferrin N-lobe (N-oTf) to anion/chelators has been resolved using kinetic and mechanistic approach. The iron release kinetics of N-oTf were measured at the endosomal pH of 5.6 with three different anions such as nitrilotriacetate, pyrophosphate, and sulfate using stopped flow spectrofluorimetric method, all yielding clear biphasic progress curves. The two observed rate constants and the corresponding amplitudes obtained from the double exponential curve fit to the biphasic curves varied depending on the type and concentration of anions. Several possible models for the iron release kinetic mechanism were examined on the basis of a newly introduced quantitative equation. Results from the curve fitting analyses were consistent with a dual pathway mechanism that includes the competitive iron release from two different protein states, namely, X and Y, with the respective first order rate constants of K(1) and K(2) (X, domain closed holo N-oTf; Y, anion induced different conformer of holo N-oTf). The reversible interconversions of X to Y and Y to X are driven by the second order rate constant k(3) and the first order rate constant K(4), respectively. The obtained rate constants were greatly variable for the three anions depending on the synergistic or nonsynergistic nature. In the light of the anion-binding sites of N-oTf located crystallographically, the compatible mechanistic model that includes competitive anion binding to the iron coordination sites and to a specific anion site is suggested for the dual pathway iron release mechanism.     

Doxorubicin and beta-lapachone release and interaction with micellar core materials: experiment and modeling

Polymer micelles with two different core-forming blocks, poly(d,l -lactide) (PLA) and poly(epsilon-caprolactone) (PCL), but the same coronal material, poly(ethylene glycol) (PEG), were investigated in this study as nanoscopic drug carriers. The release of two different drugs, doxorubicin (DOX) and beta-lapachone (beta-lap), from PEG(5k)-b-PCL(5k) and PEG(5k)-b-PLA(5k) micelles was studied at pH 5.0 and 7.4. Mathematical solutions of both Higuchi's model and Fickian diffusion equations were utilized to elucidate the differences between the micelle core materials for the two drugs. The neutral and smaller of the two drugs tested, beta-lap, demonstrated faster, pH-independent release, suggesting that no substantial changes occurred in either micelle core at lower pH. In contrast, the release rate of DOX was found to noticeably increase at lower pH with a larger cumulative amount of drug released. Different core materials were shown to have considerable influence on the release kinetics of both drugs: in both cases, the more hydrophobic PCL core showed slower drug release rates compared with the less hydrophobic PLA core.     

Season variability of iron effects on periodic culture of microalgae Dunaliella viridis Teod.

Effects of different iron concentrations (final concentrations of iron in Artari's medium: 3.7, 37.0, 74.0, and 185.0mmol·L-1) on growth rate and contents of protein, triacylglycerides, and β-carotene in Dunaliella viridis cells at cultivation in different months were investigated. It was shown that the dose-dependent effects of iron were notable for season variability. In the 1stexperimental series (October, 2007), iron at researched concentrations did not affect growth rate of culture and protein, triacylglyceride, and [3-carotene contents in cells. In experimental series conducted respectively in Novem-ber 2007, December 2007, and February 2008, the dose-dependent stimulation of microalgae growth was observed. For each of these experimental series, there were particular dose dependences of protein, triacylglyceride, and β-carotene contents in microalgae cells at cultivation on media with iron at different concentrations. Meanwhile, for all of the four experimental series conducted in different months, variability of growth rate and analyzed parameters of microalgae Dunaliella viridis as control (cultivation without iron) was shown. It is suggested that these functional differences of control cultures of microalgae in different months caused variability in the dose-dependent effects of iron in a Dunaliella viridis culture. The possibility of iron usage for increasing microalgae biomass and for enriching it by β-carotene in Dunaliella viridis culture with initial low productivity and low β-carotene content is considered.     

电化学技术研究铁蛋白接受电子的能力

Ｈｏｎｇ鱼肝脏铁蛋白（Ｌｉｖｅｒ Ｆｅｒｒｉｔｉｎ ｏｆ Ｄａｓｙａｔｉｓ Ａｋａｊｅｉ,ＤＡＬＦ）利用自身的电子隧道直接从铂金电极上获得还原电子且用于释放铁反应。血红素不仅能络合于ＤＡＬＦ,形成ＤＡＬＦ－ｈｅｍｅ分子（ＤＡＬＦＨ）,并构建成电子隧道－血红素结构,而且加速ＤＡＬＦＨ从铂金电极上接受电子的速率,从而达到提高释放铁速率的效果。用抗环血酸作为还原剂时,ＤＡＬＦ和ＤＡＬＦＨ释放铁速率几乎相     

Human platelets mediate iron release from transferrin by adenine nucleotide-dependent and -independent mechanisms

We assessed the ability of platelet sonicates and mediators secreted by unstimulated and thrombin-stimulated platelets to facilitate the release of iron from transferrin. Platelet sonicates and platelet conditioned media potentiated the release of iron from transferrin. The rate of release of iron was dependent on the pH of the reaction and amount of platelet sample added. Conditioned media from thrombin-stimulated platelets was more effective in mediating the release of iron from transferrin than was conditioned media from unstimulated cells. The rate of iron released from transferrin following addition of ATP and ADP in amounts equivalent to that present in platelet conditioned media was significantly less than the rate of iron released following the addition of conditioned media from platelets. Depletion of ATP and ADP in platelet conditioned media by incubation with apyrase only partially inhibited their ability to enhance the rate of iron release from transferrin. These observations indicate that platelets enhance the release of iron from transferrin by adenine nucleotide-dependent and -independent mechanisms. These observations are consistent with the hypothesis that platelets promote oxidant-induced tissue injury at sights of inflammation secondary to their ability to enhance the local release of iron from transferrin.     

The unusual co-assembly of H- and M-chains in the ferritin molecule from the Antarctic teleosts Trematomus bernacchii and Trematomus newnesi

Ferritins from the liver and spleen of the cold-adapted Antarctic teleosts Trematomus bernacchii and Trematomus newnesi have been isolated and characterized. Interestingly, only H- and M-chains are expressed and no L-chains. The H-chains contain the conserved ferroxidase center residues while M-chains harbor both the ferroxidase center and the micelle nucleation site ligands. Ferritins have an organ-specific subunit composition, they are: M homopolymers in spleen and H/M heteropolymers in liver. The M-chain homopolymer mineralizes iron at higher rate with respect to the H/M heteropolymer, which however is endowed with a lower activation energy for the iron incorporation process, indicative of a higher local flexibility. These findings and available literature data on ferritin expression in fish point to the role of tissue-specific expression of different chains in modulating the iron oxidation/mineralization process.     

The nature of iron released by resident and stimulated mouse peritoneal macrophages

Mouse peritoneal macrophages were allowed to ingest ⁵⁹Fe, ¹²⁵I-labelled transferrin-antitransferrin immune complexes, and the release of ⁵⁹Fe and degraded transferrin was studied. Some iron was released as ferritin, but a major portion was bound by bovine transferrin present in the culture medium, which contained fetal calf serum. If the medium was saturated with iron prior to incubation with the cells, little of the released iron was then bound by transferrin but appeared either as a high molecular weight fraction or, if nitrilotriacetate was present in the medium, some also appeared as a low molecular weight fraction. The release of non-ferritin iron was biphasic, the early, rapid phase being more prolonged with resident cells than with stimulated cells. The rate of release in the late phase did not differ significantly between resident and stimulated cells. Incubation at 0°C completely suppressed the release of degraded transferrin, but iron release continued at about 30% of the rate seen in control cultures at 37°C. A model for the intracellular handling of ingested iron is proposed to take account of the different release patterns of resident and stimulated macrophages.     

Reduction and release of ferritin iron by plant phenolics

The reductive release of ferritin iron by several naturally occurring o-diphenols was studied. The initial rate of iron release was quantified by spectrophotometric measurement of the Fe(ferrozine)3(2+) complex, which absorbs maximally at 562 nm. The initial rate of iron release was dependent upon o-diphenol concentration, but not on the concentration of the chromophoric chelating agent, ferrozine, Stoichiometric measurements resulted in a ratio of 2Fe(II) released per molecule of o-diphenol. The series of o-diphenols studied included, caffeic acid, chlorogenic acid, dihydrocaffeic acid, 3,4-dihydroxybenzoic acid, and several analogs. These reductants represent an oxidation reduction potential range of 0.38 volts. A direct correlation between reducing power of the o-diphenols and rate of ferritin iron release was observed. Superoxide dismutase, catalase, mannitol, or general radical traps had no effect on the rate of iron removal; however, EDTA and oxalate inhibited iron release. A mechanism for ferritin iron reduction and release by o-diphenols consistent with the experimental observations is discussed.     

Season variability of iron effects on periodic culture of microalgae Dunaliella viridis Teod.

Effects of different iron concentrations (final concentrations of iron in Artari’s medium: 3.7, 37.0, 74.0, and 185.0 mmol·L⁻¹) on growth rate and contents of protein, triacylglycerides, and β-carotene in Dunaliella viridis cells at cultivation in different months were investigated. It was shown that the dose-dependent effects of iron were notable for season variability. In the 1^st experimental series (October, 2007), iron at researched concentrations did not affect growth rate of culture and protein, triacylglyceride, and β-carotene contents in cells. In experimental series conducted respectively in November 2007, December 2007, and February 2008, the dose-dependent stimulation of microalgae growth was observed. For each of these experimental series, there were particular dose dependences of protein, triacylglyceride, and β-carotene contents in microalgae cells at cultivation on media with iron at different concentrations. Meanwhile, for all of the four experimental series conducted in different months, variability of growth rate and analyzed parameters of microalgae Dunaliella viridis as control (cultivation without iron) was shown. It is suggested that these functional differences of control cultures of microalgae in different months caused variability in the dose-dependent effects of iron in a Dunaliella viridis culture. The possibility of iron usage for increasing microalgae biomass and for enriching it by β-carotene in Dunaliella viridis culture with initial low productivity and low β-carotene content is considered.     

Iron uptake from transferrin and transferrin endocytic cycle in Friend erythroleukemia cells

Several aspects of iron metabolism were studied in cultured Friend erythroleukemia cells before and after induction of hemoglobin synthesis by dimethyl sulfoxide. The maximal rate of iron uptake from 59Fe-labeled transferrin, 1.5 X 10(6) atoms of Fe/cell per 30 min in uninduced cells, increased to 3 X 10(6) atoms/cell after 5 days of induction. The increase in iron uptake was not accompanied by a proportional increase in the number of transferrin receptors detected by 125I-labeled transferrin binding, suggesting a more efficient iron uptake by transferrin receptors in induced cells, with the rate of about 26 iron atoms per receptor per hour, compared to 15 atoms in uninduced cells. In agreement with this conclusion are results of the study of cellular 125I or 59Fe labeled transferrin kinetics. In the induced cells transferrin endocytosis and release proceeded with identical rates and all the endocytosed iron was retained inside the cell. On the other hand, transferrin release by uninduced cells was significantly slower and a substantial part of internalized 59Fe was released. On the basis of these results, different efficiency of iron release from internalized transferrin, accompanied by changes in cellular transferrin kinetics, is proposed as one of the factors determining the rate of iron uptake by developing erythroid cells.     

Superoxide ion as a primary reductant in ascorbate-mediated ferritin iron release

The mechanism of ascorbate-promoted ferritin iron reduction under aerobic conditions was studied. The initial rate of ferritin iron release was determined by spectrophotometric measurement of the Fe(ferrozine)3(2+) complex which absorbs at 562 nm. Variation of the initial ferrozine concentration had no influence on the rate of iron release suggesting that ferrozine does not participate in the rate-determining step. Experimental measurements of the initial rate of iron release as a function of ascorbate concentration resulted in saturation kinetics with Vmax = 2.0 X 10(-7) M.min-1 and KM = 1.3 X 10(-3) M. The effect of pH was quite pronounced with a maximal rate of iron release at pH 7.0. Stoichiometric measurements on the reaction mixture, with added catalase, resulted in a ratio of 2 Fe(II) released per ascorbate. Ascorbate-mediated iron release was inhibited 85% by superoxide dismutase, but 0% inhibition was noted with aposuperoxide dismutase. It is proposed that superoxide ion, generated during the iron-promoted oxidation of ascorbate, acts as a reductant of ferritin iron. A mechanism of ferritin iron release consistent with these experimental observations is discussed.     

Iron metabolism in BeWo chorion carcinoma cells. Transferrin-mediated uptake and release of iron

Growing human choriocarcinoma BeWo b24 cells contain 1.5 X 10(6) functional cell surface transferrin binding sites and 2.0 X 10(6) intracellular binding sites. These cells rapidly accumulate iron at a rate of 360,000 iron atoms/min/cell. During iron uptake the transferrin and its receptor recycle at least each 19 min. The accumulated iron is released from the BeWo cells at a considerable rate. The time required to release 50% of previously accumulated iron into the extracellular medium is 30 h. This release process is cell line-specific as HeLa cells release very little if any iron. The release of iron by BeWo cells is stimulated by exogenous chelators such as apotransferrin, diethylenetriaminepenta-acetic acid, desferral, and apolactoferrin. The time required to release 50% of the previously accumulated iron into medium supplemented with chelator is 15 h. In the absence of added chelators iron is released as a low molecular weight complex, whereas in the presence of chelator the iron is found complexed to the chelator. Uptake of iron is inhibited by 250 microM primaquine or 2.5 microM monensin. However, the release of iron is not inhibited by these drugs. Intracellular iron is stored bound to ferritin. A model for the release of iron by BeWo cells and its implication for transplacental iron transport is discussed.     

Sulfide is an efficient iron releasing agent for mammalian ferritins

The most prominent role of mammalian ferritins is to provide an extensive iron-buffering capacity to cells. The large ferritin iron stores can be mobilized in vitro, but the functional relevance of the most efficient iron releasing agents remains elusive. Sulfide is a strongly reducing chemical generated by a series of enzymes. In the presence of limited amounts of sulfide a continuous rate of iron release from ferritin was observed and a majority of the protein iron core was recovered in solution. The rate constants for iron efflux triggered by several reducing or chelating compounds have been measured and compared. Although not as efficient as reduced flavins, sulfide displayed kinetic parameters which suggest a potential physiological role for the chalcogenide in converting the iron storage protein into apoferritin. To further probe the relevance of sulfide in the mobilization of iron, several enzymes, such as NifS, rhodanese, or sulfite reductase generating reduced forms of sulfur by different mechanisms, have been assayed for their ability to catalyze the release of iron from ferritin. The results show that full reduction of sulfur into sulfide is needed to deplete iron from ferritin. These reactions suggest links between sulfur metabolism and intracellular iron homeostasis.     

Kinetics of pyrophosphate induced iron release from diferric ovotransferrin

The kinetics of pyrophosphate-induced iron release from diferric ovotransferrin were studied spectrophotometrically at 37 degrees C in 0.1 M HEPES, pH 7.0. At high pyrophosphate concentrations, the kinetics are biphasic, indicating that the rates of iron release from the two, presumably noninteracting iron-binding sites of ovotransferrin are different. The pseudo-first-order rate constants for iron release from both the fast and slow sites exhibit a hyperbolic dependence on pyrophosphate concentrations. The data suggest that pyrophosphate forms complexes with the two iron-binding sites of ovotransferrin prior to iron removal. The stability constants of the complex formed with the fast site (Keqf) and slow site (Keqs) are 8.3 M-1 and 40.4 M-1, respectively. The first-order rate constants for the dissociation of ferric-pyrophosphate from the fast site (k2f) and the slow site (k2s) are 0.062 and 0.0044 min-1, respectively. Results from urea gel electrophoresis studies suggest that iron is released at a much faster rate from the N-terminal binding site of ovotransferrin. At high pyrophosphate concentration, only C-monoferric-ovotransferrin is detected during the course of iron release. At low pyrophosphate concentration, however, a detectable amount of N-monoferric-ovotransferrin is accumulated. This result is consistent with the kinetic finding that the site with a higher k2 (0.062 min-1) has a lower affinity toward pyrophosphate (Keq = 8.3 M-1) whereas the site with a lower k2 (0.0044 min-1) has a higher affinity for pyrophosphate (Keq = 40.4 M-1).     

The iron regulatory hormone hepcidin reduces ferroportin 1 content and iron release in H9C2 cardiomyocytes

Iron plays a key pathophysiological role in a number of cardiac diseases. Studies on the mechanisms of heart iron homeostasis are therefore crucial for understanding the causes of excessive heart iron. In addition to iron uptake, cellular iron balance in the heart also depends on iron export. We provided evidence for the existence of iron exporter ferroportin 1 (Fpn1) in the heart in a recent study. The presence of hepcidin, a recently discovered iron regulatory hormone, was also confirmed in the heart recently. Based on these findings and the inhibiting role of hepcidin on Fpn1 in other tissues, we speculated that hepcidin might be able to bind with, internalize and degrade Fpn1 and then decrease iron export in heart cells, leading to an abnormal increase in heart iron and iron mediated cell injury. We therefore investigated the effects of hepcidin on the contents of Fpn1 and iron release in H9C2 cardiomyocyte cell line. We demonstrated that hepcidin has the ability to reduce Fpn1 content as well as iron release in this cell. The similar regulation patterns of hepcidin on the Fpn1 and iron release suggested that the decreased iron release resulted from the decreased content of Fpn1 induced by hepcidin. We also found that hepcidin has no significant effects on ceruloplasmin (CP) and hephaestin (Heph) — two proteins required for iron release from mammalian cells. The data imply that Fpn1, rather than Heph and CP, is the limited factor in the regulation of iron release from heart cells under physiological conditions.